Hershey and chase

DNA as the genetic material

1. HERSHEY–CHASE EXPERIMENT Dr. P. Samuel Assistant Professor, Department of Biotechnology, ANJA College, Sivakasi

2. Introduction The  Hershey–Chase experiments  were a series of  experiments  conducted in 1952  by  Alfred Hershey  and  Martha Chase  that helped to confirm that  DNA  is  genetic material . While DNA had been known to  biologists  since 1869, many scientists still assumed at the time that  proteins  carried the information for inheritance because DNA appeared simpler than proteins. In their experiments, Hershey and Chase showed that when  bacteriophages , which are composed of DNA and protein, infect bacteria, their DNA enters the host bacterial cell, but most of their protein does not. Although the results were not conclusive, and Hershey and Chase were cautious in their interpretation, previous, contemporaneous, and subsequent discoveries all served to prove that DNA is the hereditary material. Hershey shared the 1969  Nobel Prize in Physiology or Medicine  with  Max Delbrück  and  Salvador Luria  for their “discoveries concerning the genetic structure of viruses .”

3. Overview of experiment and observations

4. Historical background In the early twentieth century, biologists thought that proteins carried genetic information. This was based on the belief that proteins were more complex than DNA. Phoebus Levene's  influential "tetranucleotide hypothesis", which incorrectly proposed that DNA was a repeating set of identical  nucleotides , supported this conclusion.   The results of the  Avery–MacLeod–McCarty experiment , published in 1944, suggested that DNA was the genetic material, but there was still some hesitation within the general scientific community to accept this, which set the stage for the Hershey–Chase experiment. Hershey and Chase, along with others who had done related experiments, confirmed that DNA was the biomolecule that carried genetic information.   Before that,  Oswald Avery ,  Colin MacLeod , and  Maclyn McCarty  had shown that DNA led to the transformation of one strain of  Streptococcus pneumoniae  to another that was more  virulent . The results of these experiments provided evidence that DNA was the biomolecule that carried genetic information.

5. Methods and results Hershey and Chase needed to be able to examine different parts of the phages they were studying separately, so they needed to isolate the phage subsections. Viruses were known to be composed of a protein shell and DNA, so they chose to uniquely label each with a different elemental  isotope . This allowed each to be observed and analyzed separately. Since phosphorus is contained in DNA but not amino acids,  radioactive  phosphorus-32 was used to label the DNA contained in the  T2 phage . Radioactive sulfur-35 was used to label the protein sections of the T2 phage, because sulfur is contained in amino acids but not DNA.

6. Structure of a T2 Phage

7. Hershey and Chase inserted the radioactive elements into the bacteriophages by adding the isotopes to separate media within which bacteria were allowed to grow for 4 hours before bacteriophage introduction. When the bacteriophages infected the bacteria, the  progeny contained the radioactive isotopes in their structures. This procedure was performed once for the sulfur-labeled phages and once for phosphorus-labeled phages. The labeled progeny were then allowed to infect unlabeled bacteria. The phage coats remained on the outside of the bacteria, while genetic material entered. Disruption of phage from the bacteria by agitation in a blender followed by centrifugation allowed for the separation of the phage coats from the bacteria. These bacteria were lysed to release phage progeny. The progeny of the phages that were originally labeled with  32 P remained labeled, while the progeny of the phages originally labeled with  35 S were unlabeled. Thus, the Hershey–Chase experiment helped confirm that DNA, not protein, is the genetic material.

8. Hershey and Chase showed that the introduction of  deoxyribonuclease  (referred to as  DNase ), an  enzyme  that breaks down DNA, into a solution containing the labeled bacteriophages did not introduce any  32 P into the solution. This demonstrated that the phage is resistant to the enzyme while intact. Additionally , they were able to  plasmolyze  the bacteriophages so that they went into osmotic shock, which effectively created a solution containing most of the  32 P and a heavier solution containing structures called “ghosts” that contained the  35 S and the protein coat of the virus. It was found that these “ghosts” could  adsorb  to bacteria that were susceptible to T2, although they contained no DNA and were simply the remains of the original bacterial capsule. They concluded that the protein protected the DNA from DNAse, but that once the two were separated and the phage was inactivated, the DNAse could hydrolyze the phage DNA.  However, it subsequently became clear that in  some viruses , RNA is the genetic material.

9. Experiment and conclusions Hershey and Chase were also able to prove that the DNA from the phage is inserted into the bacteria shortly after the virus attaches to its host. Using a high speed blender they were able to force the bacteriophages from the bacterial cells after  adsorption . The lack of  32 P labeled DNA remaining in the solution after the bacteriophages had been allowed to adsorb to the bacteria showed that the phage DNA was transferred into the bacterial cell. The presence of almost all the radioactive  35 S in the solution showed that the protein coat that protects the DNA before adsorption stayed outside the cell . Hershey and Chase concluded that DNA, not protein, was the genetic material. They determined that a protective protein coat was formed around the bacteriophage, but that the internal DNA is what conferred its ability to produce progeny inside a bacterium. They showed that, in growth, protein has no function, while DNA has some function. They determined this from the amount of radioactive material remaining outside of the cell. Only 20% of the  32 P remained outside the cell, demonstrating that it was incorporated with DNA in the cell's genetic material. All of the  35 S in the protein coats remained outside the cell, showing it was not incorporated into the cell, and that protein is not the genetic material . Hershey and Chase's experiment concluded that little sulfur containing material entered the bacterial cell. However no specific conclusions can be made regarding whether material that is sulfur-free enters the bacterial cell after phage adsorption. Further research was necessary to conclude that it was solely bacteriophages' DNA that entered the cell and not a combination of protein and DNA where the protein did not contain any sulfur.

10. Discussion Confirmation Hershey and Chase concluded that protein was not likely to be the hereditary genetic material. However, they did not make any conclusions regarding the specific function of DNA as hereditary material, and only said that it must have some undefined role . Confirmation and clarity came a year later in 1953, when  James D. Watson  and  Francis Crick  correctly hypothesized, in their journal article " Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid ", the  double helix  structure of DNA, and suggested the  copying mechanism  by which DNA functions as hereditary material. Furthermore , Watson and Crick suggested that DNA, the genetic material, is responsible for the synthesis of the thousands of proteins found in cells. They had made this proposal based on the structural similarity that exists between the two macromolecules, that is, both protein and DNA are linear sequences of amino acids and nucleotides respectively.

11. Genetic Code: Meaning, Types and Properties Meaning of Genetic Code The genetic code may be defined as the exact sequence of DNA nucleotides read as three letter words or codons, that determines the sequence of amino acids in protein synthesis. In other words, the genetic code is the set of rules by which information encoded in genetic material (DNA or RNA sequences) is translated into proteins (amino acid sequences) by living cells.

12. The main points related to genetic code The genetic code is ‘read’ in triplets of bases called codons. In other words, a set of three nucleotide bases constitutes a codon . In a triplet code, three RNA bases code for one amino acid . There are 64 codons which correspond to 20 amino acids and to signals for the initiation and termination of transcription . The code uses codons to make the amino acids that, in turn, constitute proteins.

13. Each triplet [codon] specifies one amino acid in a protein structure or a start signal or stop signal in protein synthesis. The code establishes the relationship between the sequence of bases in nucleic acids (DNA and the complementary RNA) and the sequence of amino acids in proteins. The code explains the mechanism by which genetic information is stored in living organisms.

14. Types of Genetic Code The genetic code is of two types. The genetic code can be expressed as either RNA codons or DNA codons. RNA codons occur in messenger RNA (mRNA) and are the codons that are actually  “read”  during the synthesis of polypeptides (the process called translation ). But each mRNA molecule acquires its sequence of nucleotides by transcription from the corresponding gene [DNA], Because DNA sequencing has become so rapid and because most genes are now being discovered at the level of DNA before they are discovered as mRNA or as a protein product, it is extremely useful to have a table of codons expressed as DNA. Both tables are given here.

15. DNA Codons These are the codons as they are read on the sense (5′ to 3′) strand of DNA. Except that the nucleotide thymine (T) is found in place of uracil (U), they read the same as RNA codons. However, mRNA is actually synthesized using the antisense strand of DNA (3′ to 5′) as the template.

16. Types of Codon The genetic code consists of 64 triplets of nucleotides. These triplets are called codons. With three exceptions, each codon encodes for one of the 20 amino acids used in the synthesis of proteins. This produces some redundancy in the code . Most of the amino acids are encoded by more than one codon. One codon that is AUG serves two related functions. It signals the start of translation and codes for the incorporation of the amino acid methionine (Met) into the growing polypeptide chain.

17. Properties of Genetic Code The genetic code is: ( i ) Triplet, (ii) Universal, (iii) Comma-less, (iv) Non-overlapping, (v) Non-ambiguous , (vi) Redundant, and (vii) Has polarity.

18. The Code is Triplet The genetic code is triplet. The triplet code has 64 codons which are sufficient to code for 20 amino acids and also for start and stop signals in the synthesis of polypeptide chain. In a triplet code three RNA bases code for one amino acid . The Code is Universal The genetic code is almost universal. The same codons are assigned to the same amino acids and to the same START and STOP signals in the vast majority of genes in animals, plants, and microorganisms. 

19. The Code is Commaless It is believed that the genetic code is commaless . In other words, the codons are continuous and there are no demarcation lines between codons. Deletion of a single base in a commaless code alters the entire sequence of amino acids after the point of deletion as given below.

20. The Code is Non-Overlapping Three nucleotides or bases code for one amino acid. In a non-overlapping code, six bases will code for two amino acids. In a non-overlapping code, one letter is read only once. In overlapping code, six nucleotides or bases will code for 4 amino acids, because each base is read three times.

21. The Code is Non-ambiguous The genetic code has 64 codons. Out of these, 61 codons code for 20 different amino acids. However, none of the codons codes for more than one amino acid. In other words, each codon codes only for one amino acid. This clearly indicates that the genetic code is non-ambiguous. In case of ambiguous code, one codon should code for more than one amino acid. In the genetic code there is no ambiguity.

22. The Code is Redundant In most of the cases several codons code for the same amino acid. Only two amino acids, viz. tryptophan and methionine are coded by one codon each. Nine amino acids are coded by two codons each, one amino acid [ Isoleucine ] by three codons, five amino acids by 4 codons each, and three amino acids by 6 codons.

23. The Code Has Polarity The code has a definite direction for reading of message, which is referred to as polarity. Reading of codon in opposite direction will specify for another amino acid due to alteration in the base sequences in the code. In the following codons, reading of message from left to right and right to left will specify for different amino acids. Because the codon in the following case will be read as UUG from left to right and as GUU from right to left which codes for another amino acid .